Clinicopathological and biological significance of aberrant activation of glycogen synthase kinase-3 in ovarian cancer.

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ORIGINAL RESEARCH

Clinicopathological and biological significance
of aberrant activation of glycogen synthase
kinase-3 in ovarian cancer

This article was published in the following Dove Press journal:
OncoTargets and Therapy
25 June 2014

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Yunfeng Fu 1
Xinyu Wang 1
Xiaodong Cheng 1
Feng Ye 2
Xing Xie 12
Weiguo Lu 12

'Department of Gynecologic
Oncology, Women's Hospital, School
of Medicine, Zhejiang University,
Women's Reproduction and Health
Laboratory of Zhejiang Province,
Hangzhou, People's Republic of China

Correspondence: Weiguo Lu
Department of Gynecologic Oncology,
Women's Hospital, School of Medicine,
Zhejiang University, Xueshi Road #2,
Hangzhou 310006, People's Republic
of China

Tel +86 571 8706 1 50 1 ext 1005

Fax +86 571 8706 1878

Email lbwg@zju.edu.cn; lwg@hzcnc.com

Background: Glycogen synthase kinase-3 (GSK-3) plays an important role in human cancer.
The aim of this study is to evaluate the clinicopathological significance of expression of GSK-
3oc/p and pGSK-3oc/p Tyr279/216 in patients with epithelial ovarian cancer and to investigate whether
GSK-3 inhibition can influence cell viability and tumor growth of ovarian cancer.
Methods: Immunohistochemistry was used to examine expression of GSK-3a/p andpGSK-3a/
pTyr279/2i6 j n 7 1 human epithelial ovarian cancer tissues and correlations between protein expres-
sion, and clinicopathological factors were analyzed. Cell viability was determined by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay following exposure of
ovarian carcinoma cells to pharmacological inhibitors of GSK-3 or GSK-3 small interfering
RNA. In vivo validation of tumor growth inhibition was performed with xenograft mice.
Results: The expression levels of GSK-3a/(3 and pGSK-3a/(3 Tyr279 ' 216 in ovarian cancers were
significantly higher than those in benign tumors. High expression of GSK-3a/(3 was more
likely to be found in patients with advanced International Federation of Gynecology and
Obstetrics (FIGO) stages and high serum cancer antigen 125. Higher expression of pGSK-3oc/
pTyr279/2i6 was ass0 ciated with advanced FIGO stages, residual tumor mass, high serum cancer
antigen 125, and poor chemoresponse. Worse overall survival was revealed by Kaplan-Meier
survival curves in patients with high expression of GSK-3a/p or pGSK-3a/p Tyr279/216 . Multivariate
analysis indicated that FIGO stage, GSK-3a/p expression, and pGSK-3a/p Tyr27 " 216 expression
were independent prognostic factors for overall survival. GSK-3 inhibition by lithium chloride,

4- benzyl-2-methyl-l,2,4-thiadiazolidine-3,5-dione (TDZD-8), or GSK-3 small interfering RNA
can decrease viability of SKOV3 and SKOV3-TR30 ovarian cancer cells. Additionally, lithium
chloride-treated SKOV3 xenograft mice had a significant reduction in tumor growth compared
with control-treated animals.

Conclusion: Our findings suggest that overexpression and aberrant activation of GSK-3 may
contribute to progression and poor prognosis in ovarian cancer. Inhibition of GSK-3 may be a
potential therapy for ovarian cancer.

Keywords: ovarian carcinoma, immunohistochemistry, lithium chloride, TDZD-8

Introduction

Despite the skillful surgical techniques and improvements in combined chemotherapy,
ovarian cancer remains the leading cause of death in gynecological malignancies with

5- year survival rates of 44%.' The main reason for poor prognosis of ovarian cancer
is that about 70% of the cases are still diagnosed in advanced stages. The majority of
advanced patients, including those who achieve a complete response to first-line chemo-
therapy, will ultimately relapse and do not successfully respond to further treatments. 2

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I 159

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There is clearly an urgent need to develop new classes
of treatment modalities, represented by molecular target-
directed therapies.

Glycogen synthase kinase-3 (GSK-3) is a multifunctional
serine (Ser)/threonine kinase known to play a pivotal role in
the regulation of metabolism, embryonic development, cell
differentiation, and apoptosis. 34 Two isoforms of GSK-3,
GSK-3a and GSK-3p, have been identified with high homol-
ogy and similar but not identical biochemical functions. The
actions of GSK-3 are often regulated by phosphorylation.
In opposition to inhibitory regulation by phosphorylation of
Ser9 in GSK-3|3 and Ser21 in GSK-3a, GSK-3 activity is
enhanced by phosphorylation of tyrosine (Tyr)2 1 6 in GSK-3 (3
andTyr279 in GSK-3a. 4 Surprisingly, most of the research so
far has primarily focused on GSK-3 (3 only. The dysregulation
of GSK-3 (3 has been implicated in tumorigenesis and cancer
progression. 5 It remains controversial whether GSK-3 (3 is a
"tumor promoter" or "tumor suppressor". 5,6

Our interest in GSK-3 stemmed from our previous study
in which we found that enhanced expression of GSK-3 is
associated with acquired resistance to paclitaxel in ovarian
carcinoma cells. 7 Previous studies have investigated the effect
of GSK-3(3 inhibition in ovarian cancer cells. 8-11 However,
the clinicopathological and biological significance of GSK-3
in ovarian cancer remains inconclusive.

In the present study, we extended the previous study to
evaluate the expression status of GSK-3a/(3 and its actively
phosphorylated form, pGSK-3a/p Tyr279/216 , in ovarian cancer
tissues using immunohistochemical analysis. 7 The associa-
tions between clinicopathological factors and expression of
GSK-3a/(3 and pGSK-3a/p Tyr279/216 were evaluated. In addi-
tion, we provide experimental evidence that GSK-3 inhibition
by pharmacological agents or RNA interference can decrease
viability of ovarian carcinoma cells and slow tumor growth
in vivo.

Materials and methods

Clinical data and tissue samples

This study included 7 1 patients with epithelial ovarian cancer
(EOC) who underwent surgical resection followed by pacli-
taxel/platinum combined chemotherapy in Women's Hospital,
School of Medicine, Zhejiang University, in the period 2003
to 2005. The paraffin-embedded specimens were retrieved
from the archives of the Department of Pathology of our
hospital. Meanwhile, we also collected 20 samples of benign
ovarian tumor (ten serous and ten mucinous cystadenoma)
as controls. The use of all tissues has been approved by the
Ethics Committee of Women's Hospital, School of Medicine,

Zhejiang University (No 20100009). Because the use of
redundant paraffin-embedded specimens did not bring any
damage to patients, the Ethics Committee waived the need
for consent from the patients. All patients with EOC were
followed-up two to four times annually until December 2010.
The following data were collected: age, date of diagnosis, date
of relapse, date of last follow-up, date and cause of death,
serum cancer antigen 125 (CA125) values, International
Federation of Gynecology and Obstetrics (FIGO) stage,
histologic type and grade, residual tumor mass, and response
to chemotherapy. Chemotherapeutic response was evalu-
ated according to the Response Evaluation Criteria in Solid
Tumors (RECIST). 12 Patients achieving complete response or
partial response to treatment were considered as responders,
and patients achieving progressive disease or stable disease
were considered as nonresponders.

Immunohistochemistry

Expression of GSK-3a/(3 and pGSK-3a/p Tyr279/216 in tissues
was examined immunohistochemically by the avidin-biotin-
peroxidase complex method following microwave antigen
retrieval of paraffin sections, using a mouse monoclonal
antibody to human GSK-3a/p (diluted 1:30; Santa Cruz
Biotechnology Inc., Dallas, TX, USA) and a rabbit polyclonal
antibody to human pGSK-3a/p Tyr279/216 (diluted 1:80; Santa
Cruz Biotechnology), respectively. The expression levels of
GSK-3a/p and pGSK-3a/p Tyr279/216 were determined semi-
quantitatively according to the described method. 13 Briefly,
the percentage of positive cells was rated as follows: 0 points,
0% to 25%; 1 point, 26% to 50%; 2 points, 51% to 75%;
3 points, >75%. The staining intensity was rated as follows:

0 points, no staining; 1 point, weak intensity; 2 points, moder-
ate intensity; 3 points, strong intensity. Points for expression
intensity and percentage of positive cells were added (total
score of 0-6). Thus, the score 0 was taken as negative (-);

1 to 2 as weak positive (+); 3 to 4 as moderate positive (++);
and 5 to 6 as strong positive (+++).

Cell culture and reagents

Human ovarian carcinoma cell line SKOV3 was obtained from
the American Type Culture Collection (Manassas, VA, USA)
and maintained in McCoy's 5A medium (Gibco®; Thermo
Fisher Scientific, Waltham, MA, USA), supplemented with
10% fetal bovine serum (FBS), 100 U/mL penicillin, and
100 ng/mL streptomycin at 37°C and 5% C0 2 . Paclitaxel-
resistant cell subline, SKOV3-TR30, was developed by
exposing parental SKOV3 cells to increased concentration
of paclitaxel as described previously. 14 Two GSK-3 inhibi-

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Aberrant activation of GSK-3 in ovarian cancer

tors, lithium chloride (LiCl) and 4-benzyl-2 -methyl- 1,2,4-
thiadiazolidine-3,5-dione (TDZD-8), were purchased from
Sigma-Aldrich (St Louis, MO, USA).

Cell viability assay

The effect of LiCl and TDZD-8 on cell viability was deter-
mined by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT) assay as described previously. 14 Briefly,
2,000 cells per well were seeded in 96-well plates, incubated
in a C0 2 incubator overnight at 37°C, and then treated with
different doses of LiCl (0-80 mM) and TDZD-8 (0-20 [iM)
in triplicate for indicated times. A volume of 10 |lL of MTT
(5 mg/mL) was added to the cells and incubated at 37°C
for 3 hours. The MTT solution was removed and 200 |iL of
dimethyl sulfoxide was added to each well to dissolve the
blue formazan crystals. The optical density was measured
at 490 nm wavelength using a Universal microplate reader
Elx800 (BIO-TEK, Winooski, VT, USA). The absorbance
values were normalized by assigning the value of the control
line in the medium without drug to 1.0 and the value of the
no-cell control to 0. Mean values were calculated from three
independent experiments.

RNA interference

Small interfering RNA (siRNA) specific to human GSK3oc
(GSK-3oc siRNA [h] sc-29339) and GSK3(3 (GSK-3(3
siRNA [h] sc-35527) were purchased from Santa Cruz
Biotechnology. Unrelated control siRNA (sc-37007; Santa
Cruz Biotechnology) was also used. Transfection was car-
ried out using DharmaFECT siRNA transfection reagents
(Thermo Fisher Scientific) according to the manufacturer's
recommendations. Briefly, 24 hours after plating, cells were
incubated with the siRNA/DharmaFECT complexes at the
appropriate concentration (70 nM for GSK3a, 50 nM for
GSK3(3) in antibiotic- and serum- free medium for 6 hours
at 37°C. After this incubation period, transfection medium
was replaced with fresh medium containing FBS. The effect
of siRNA on protein expression was determined by Western
immunoblotting, and the relative cell viability was deter-
mined by MTT assay 72 hours after transfection.

Western blotting

Cells were harvested and lysed in modified protein lysis
buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1%
sodium dodecyl sulfate [SDS], 1% NP-40, 0.02% sodium
azide) added with 1% proteinase inhibitor cocktail (Sigma-
Aldrich). The protein concentration was measured by the
Bradford method. 15 Equal amounts of sample lysates were

separated by SDS-PAGE and electrophoretically trans-
ferred onto a nitrocellulose membrane. The membrane was
blocked with 5% nonfat dried milk in TBST buffer (20 mM
Tri-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween-20) and
incubated overnight at 4°C with the antibodies against
GSK-3a/[3 (1:500 mouse monoclonal antibody) and
P-actin (1:1 ,000 mouse monoclonal antibody; Santa Cruz
Biotechnology), respectively. The membrane was washed
with TBST buffer and incubated with appropriate second-
ary antibodies. The protein bands were visualized using the
enhanced Pierce chemiluminescence kits (Thermo Fisher
Scientific). Signal data was normalized for P-actin bands,
and a mean value was calculated from three independent
experiments.

Nude mice and xenografts

Eight female BALB/c-nude mice, aged 4-5 weeks, were pur-
chased from Shanghai Laboratory Animal Center (Shanghai,
People's Republic of China) and housed within a dedicated
specific pathogen- free facility at the Laboratory Animal Cen-
ter of Zhejiang University. To develop the tumor xenografts,
5xl0 6 SKOV3 cells (in 0.2 mL PBS) were injected subcutane-
ously near the axillary fossa of nude mice. Once tumors were
palpable, mice were randomly divided into two groups (four
mice per group) and treated with various regimens: normal
saline control or LiCl. LiCl (340 mg/kg, intraperitoneally)
or normal saline were injected every 2 days for a total of
seven treatments (day 1, 3, 5, 7, 9, 11, 13). Length (1) and
width (w) of tumors were measured with a vernier caliper
once a week, and the tumor volumes were calculated using
the formula,

0.52xlxw 2 . (1)

At the end of the study (7 weeks after drug treatment),
animals were sacrificed according to the protocol developed
by the Guidance of the Animal Center, and tumors were
excised and weighed.

Statistical analysis

The data were compiled with the software package SPSS,
version 16.0 (Chicago, IL, USA). Mann-Whitney U tests
were used to assess the correlation between clinicopathologi-
cal parameters and expressions of GSK-3a/p andpGSK-3a/
pTyr279/2i6 Univariate survival analysis was performed by
the Kaplan-Meier method (log-rank test). The multivariate
survival analysis was performed according to the Cox regres-
sion model. Differences in data among treatment groups in
experiments in vitro and in vivo were analyzed using one-way

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analysis of variance (ANOVA). All tests were two-tailed, and
P<0.05 was considered to be significant.

Results

Increased expression of GSK-3a/(3
and pGSK-3a/(3 T y r279/216 in ovarian
cancer tissues

Using immunohistochemistry, we investigated the expres-
sion of GSK-3a/(3 and pGSK-3a/p Tyr279/216 in specimens
of EOC and benign tumors. Figures 1 and 2 show the
representative features of immunohistochemical staining
of GSK-3oc/p and pGSK-3a/p Tyr279/216 , respectively. The
expression levels of GSK-3a/(3 and pGSK-3a/p Tyr279/216 in
EOC were significantly higher than those in benign tumors
(.P<0.001) (Tables 1 and 2). Moreover, a significant cor-
relation between expression of GSK-3a/(3 and pGSK-3a/
p Ty r279/2i6 was 0 b se rved (r=0.278, P=0.019). Thus, it is likely
that overexpression and aberrant activation of GSK-3 is a
pathologic characteristic of clinical ovarian cancer.

In patients with EOC, the clinicopathologic and prog-
nostic significance of expression levels of GSK-3 a/(3 and
pGSK-3a/(3 Tyr279/216 were also analyzed. High expression of

Figure I Representative features of immunohistochemical staining of GSK-3a/p.
Notes: Little staining of GSK-3a/p in benign epithelial ovarian tumor (200x)
(A) and (400x) (B); EOC case with low expression of GSK-3a/p (200x) (C)
and (400x) (D); EOC case with high expression of GSK-3a/p (200x) (E) and
(400x) (F).

Abbreviations: EOC, epithelial ovarian cancer; GSK, glycogen synthase kinase.

A

B

~~ > • l.'-'-'z.-S- ■ "~ : " -;' ' " J
m '•• •;. - •• : r.:-s?.i/>v

C ^.'.,3

D

Figure 2 Representative features of immunohistochemical staining of pGSK-3a/

pTyr279/2l6

Notes: Little staining of pGSK-3ct/p Tyr279/216 in benign epithelial ovarian tumor
(200x) (A) and (400x) (B); EOC case with low expression of pGSK-3a/p T ' r27 " 216
(200x) (C) and (400x) (D); EOCcasewith high expression of pGSK-3a/p T ' r27 " 216 (200x)
(E) and (400x) (F).

Abbreviations: EOC, epithelial ovarian cancer; GSK, glycogen synthase kinase.

GSK-3a/(3 was more likely to be found in EOC patients with
advanced FIGO stages (P=0.001) and high serum CA125
CP=0.040) (Table 1). Higher expression of pGSK-3oc/p Tyr279/216
was more prevalent in tissues from patients with advanced
FIGO stages (P=0.004), residual tumor mass (P=0.044),
high serum CA125 (P=0.003), and poor chemoresponse
(F=0.001) (Table 2). Until the final follow-up time, the
median follow-up time of the total 71 patients was 59 months
(range 5-87). Worse overall survival was revealed by Kaplan-
Meier survival curves in patients with high expression of
GSK-3a/p (P=0.004) or pGSK-3a/p Tyr279/216 (P=0.005)
(Figure 3). Multivariate analysis indicated that FIGO stage
(hazard ratio [HR] =11.1, P=0.02), GSK-3a/p expression
(HR =3.1, P=0.045), and pGSK-3a/p Tyr279/216 expression
(HR =2.8, P=0.041) were independent prognostic factors
for overall survival.

Inhibition of GSK-3 decreases viability
of ovarian carcinoma cells

The immunohistochemical results in our study indicate that
GSK-3 has a putative pathologic role in ovarian carcinoma.
To determine whether active GSK-3 is essential for survival

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Aberrant activation of GSK-3 in ovarian cancer

Table I Expression of GSK-3a/(3 in epithelial ovarian cancer and
benign ovarian tumor

Parameters GSK-3<x/p z P-value

immunostaining

- + ++ +++

Benign or malignant

-5.956

<0.00l

Benign tumor (n=20)

18

2

0

0

EOC (n=7l)

8

21

19

23

Age of EOC patient (years)

-0.222

0.824

£50 (n=38)

3

13

1 1

1 1

>50 (n=33)

5

8

8

12

Stage of EOC

-3.328

0.001

FIGO 1 + II (n=27)

5

1 1

9

2

FIGO III + IV (n=44)

3

10

10

21

Residual tumor of EOC

-1.406

0.160

£ 1 cm (n=53)

8

15

15

15

> 1 cm (n=l8)

0

6

4

8

Histological type of EOC

-0.078

0.938

Serous (n— 39)

3

14

9

13

Other (n=32)

5

7

10

10

Histological grade of EOC

-1.552

0.121

Grade 1 (n=l 1)

1

5

5

0

Grade 2 + 3 (n=60)

7

16

14

23

Serum CAI25 of EOC

-2.053

0.040

patient (U/mL)

£500 (n=47)

8

15

1 1

13

>500 (n=24)

0

6

8

10

Chemotherapy response

-1.234

0.217

of EOC

Response (n=55)

6

19

14

16

Nonresponse (n= 1 6)

2

2

5

7

Abbreviations: CA I 25, cancer antigen 1 25; EOC, epithelial ovarian cancer; FIGO,
International Federation of Gynecology and Obstetrics; GSK, glycogen synthase
kinase.

of ovarian carcinoma cells, we tested the effect of two GSK-3
inhibitors, LiCl andTDZD-8, in SKOV3 and SKOV3-TR30,
a paclitaxel-resistant cell subline derived from SKOV3. We
found that both LiCl andTDZD-8 can decrease cell viability
of SKOV3 and SKOV3-TR30 (Figure 4A-D).

To determine whether the reduction of cell viability by
pharmacological inhibition of GSK-3 was specific to GSK-3
isoforms, we depleted the expression of GSK-3a or GSK-3(3
in SKOV3 and SKOV3-TR30 cells using RNA interference.
Following a 3-day exposure to GSK-3 -specific siRNAs,
immunoblotting showed ^70% reduction in expression
levels of the corresponding GSK-3 isoforms when compared
to untransfected or scrambled siRNA transfected controls
(Figure 4E). We found that depletion of either GSK-3a or (3
isoforms can significantly decrease cell viability of ovarian
cancer (Figure 4F). These results indicate that both GSK-3
isoforms may contribute to regulate cell viability of ovarian
carcinoma.

Table 2 Expression of pGSK-3a/(3 T),r279 ' 216 in epithelial ovarian
cancer and benign ovarian tumor

Parameters pGSK-3<x/p Tyr27 " 216 z P-value

immunostaining

- + ++ +++

Benign or malignant

-4.759

<0.00l

Benign tumor (n=20)

16

2

1

1

EOC (n=7l)

1 1

22

14

24

Age of EOC patient (years)

-0.685

0.508

£50 (n=38)

8

10

8

12

>50 (n=33)

3

12

6

12

Stage of EOC

-2.8 1 5

0.004

FIGO 1 + II (n=27)

5

13

6

3

FIGO III + IV (n=44)

6

9

8

21

Residual tumor of EOC

-2.005

0.044

£ 1 cm (n=53)

10

18

10

15

> 1 cm (n= 1 8)

1

4

4

9

Histological type of EOC

-1.518

0.132

Serous (n— 39)

5

9

10

15

Other (n=32)

6

13

4

9

Histological grade of EOC

-0.298

0.791

Grade 1 (n=l 1)

1

5

2

3

Grade 2 + 3 (n=60)

10

17

12

21

Serum CAI25 of EOC

-2.920

0.003

patient (U/mL)

£500 (n=47)

10

17

9

1 1

>500 (n=24)

1

5

5

13

Chemotherapy response

-3.292

0.001

of EOC

Response (n=55)

10

20

13

12

Nonresponse (n=l6)

1

2

1

12

Abbreviations: CAI25, cancer antigen 125; EOC, epithelial ovarian cancer; FIGO,
International Federation of Gynecology and Obstetrics; GSK, glycogen synthase
kinase.

Effect of LiCl on tumor growth in vivo

To determine whether GSK-3 inhibition affects ovarian
tumor growth in vivo, we used a xenograft model of ovarian
cancer generated by SKOV3 cells. As shown in Figure 5A,
tumor growth was slowed in the LiCl group as measured
by calculated tumor volume. The differences between the
control and LiCl-treated tumor volumes at days 21, 28, and
35 were statistically significant. By the end of the study, the
differences of tumor volumes between control and treatment
group were statistically insignificant (Figure 5A and B).
However, tumor growth was significantly reduced in the
LiCl group as measured by tumor weight when tumors were
excised at the end of the study (Figure 5C).

Discussion

In view of the known biological importance of GSK-3
in human cancers and the unknown clinicopathological
significance of GSK-3 expression in ovarian carcinoma,

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LO-

TS 08
>

| 0.6-
tfl

g 0.4-

a>
>

°0.2-

o.o-

20

Low GSK-3a/(3

High GSK-3a/p

P=0.004

40 60

Months

80

100

B

1.0-

>

in

; 0.6-

2 0.4-
°0.2-

o.o-

20

Low pGSK-Sa/p 1 " 275

High pGSK-Sa/p 1 " 279 ' 216

P=0.005

40 60

Months

80

100

Figure 3 Kaplan-Meier survival analysis among 71 patients with epithelial ovarian cancer according to expression of GSK-3a/fi and pGSK-3a/p Tyr279/2ls .

Notes: Patients with high expression (+H h++) of GSK-3a/p (A) or pGSK-3a/p T>,r279/216 (B) had a significantly inferior overall survival time than those with low expression

(P=0.004, P=0.005, respectively).
Abbreviation: GSK, glycogen synthase kinase.

we used immunohistochemistry to assess the expression
status of GSK-3a/(3 and its actively phosphorylated form,
pGSK-3a/p Tyr279/216 , in a series of 71 EOC. In this study, we
performed immunohistochemical analysis using two anti-
bodies which can simultaneously recognize GSK-3a and
GSK-3[3 and the activated form of GSK-3a and GSK-3P,
respectively. We found that the expression levels of GSK-
3a/p and pGSK-3a/p Tyr279/216 in EOC were significantly
higher than those in benign ovarian tumors. High expressions
(++ - +++) of GSK-3a/p were observed in 59.2% (42/71)
of the EOC samples, which correlated with advanced FIGO

stage and high serum CA125. High expressions (-H H-+)

of pGSK-3a/p Tyr279/216 were observed in 53.5% (38/71) of
EOC, which associated with advanced FIGO stage, post-
operative residual tumor mass, and high serum C A 125. These
findings reflect that expression of GSK-3a/p and pGSK-3oc/
pTyr279/2i6 seem increase with tumor growth and invasive-
ness, and the aberrant activation of GSK-3a/p may play a
role in the progression of EOC. Consistently, Do et al found
that pGSK-3a/p Scr9/21 immunostaining in benign neoplasias
was significantly higher than in ovarian carcinomas. 16 Loss
of pGSK-3a/p Scr9/21 immunoreactivity indirectly indicates
upregulation of GSK-3 activity, despite having not detected
pGSK-3a/p Tyr279/216 immunostaining.

EOC is mainly treated by paclitaxel/platinum combined
chemotherapy, and this study has evaluated the expression of
GSK-3a/p or pGSK-3a/p Tyr279/216 as a marker of chemothera-
peutic responsiveness in such patients. In our study, 77.5%
(55/71) of EOC patients showed a response to the combined
chemotherapy, and no correlation was observed between
GSK-3a/p expression and chemotherapeutic responsiveness.
However, we found that expressions of pGSK-3a/p Tyr279/216

were clearly lower in responders than those in nonresponders.
The response rate (30/33) in patients with low expression
(- - +) of pGSK-3a/p Tyr279/216 was increased significantly
These data strongly suggest that the expression level of
activated GSK-3 in ovarian cancer is inversely associated
with responsiveness of paclitaxel/platinum chemotherapy.
High expression of pGSK-3a/p Tyr279/216 in cancer tissue may
be a potential predictor for primary resistance to the first-line
chemotherapy. Our results are inconsistent with the report
by Cai et al, 9 who demonstrated that GSK-3 P inhibition may
confer resistance to cisplatin in the A2780 ovarian carcinoma
cell line in vitro. However, their results came from only one
cell model, without verification of clinical samples. It cannot
be excluded that GSK-3 may play a different role in different
ovarian cancer cells.

There have been a number of conflicting reports concerning
the prognostic values of GSK-3P in human cancers. 17 " 23 In the
present study, we found that high expressions of GSK-3a/p
and pGSK-3a/p Tyr279/216 were significantly associated with
shortened overall survival in patients with EOC. It gave us
a clue that overexpression and aberrant activation of GSK-3
might be potential independent predictors for poor prognosis in
ovarian cancer. Our results agreed with previous results shown
in lung cancer 18 and bladder cancer. 22 Nevertheless, the cur-
rent results do not agree with those in tongue cancer, 17 breast
cancer, 19 20 gastric cancer, 21 and lymphoma. 23 Previous studies
indicated that the biological significance of GSK-3 activation
or inactivation appears to be cell-type specific. In cancers of
the pancreas, 24 colon, 25 ovary, 8 and osteosarcoma, 26 GSK-3P
activity is believed to promote cell survival or tumor growth.
Additionally, cell proliferation and survival were attenuated
by inhibiting the activity of GSK-3 P in myeloma, 27 thyroid

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Aberrant activation of GSK-3 in ovarian cancer

p 120 -

SKOV3-TR30 SKOV3

Figure 4 Effect of GSK-3 inhibition on cell viability.

Notes: LiCI (A and B) and TDZD-8 (C and D) reduces viability of ovarian carcinoma cells; SKOV3 (A and C) and SKOV3-TR30 (B and D) cells were treated with various
concentrations of LiCI or TDZD-8, and cell viability was determined using the MTT assay at 24, 48, and 72 hours; Data represent the mean of three different experiments
with triplicate wells; To determine GSK-3a/p protein levels after siRNA treatment, protein extracts were obtained using a lysis buffer containing protease inhibitors cocktail
72 hours posttransfection and analyzed by Western blot (E); A reduction of ^70% in the expression levels of the corresponding GSK-3 isoforms was observed when
compared to untransfected or scrambled siRNA transfected controls. GSK-3 silencing decreases viability of ovarian carcinoma cells (F); Cell viability was determined by
MTT assay 72 hours after transfection with GSK-3a siRNA (70 nM), GSK-3p siRNA (50 nM), or both in SKOV3 and SKOV3-TR30 cells; Data represent the mean of three
different experiments with triplicate wells. *P<0.05, # P<0.0l compared with control siRNA.

Abbreviations: GSK, glycogen synthase kinase; LiCI, lithium chloride; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; siRNA, small interfering RNA;
TDZD-8, 4-benzyl-2-methyl- 1 ,2,4-thiadiazolidine-3,5-dione.

cancer, 28 leukemia, 29 glioma, 30,31 renal cancer, 32 gastrointes-
tinal cancer, 33 and bladder cancer 22 cells. Moreover, GSK-3
inhibition may enhance the antitumor effect of TNF -related
apoptosis-inducing ligand (TRAIL) in pancreatic cancer cells
or sorafenib in renal cell carcinoma. 34 - 35 Inconsistently, GSK-3(3

seems to be a "tumor suppressor" or "proapoptotic protein"
in other cancer cells, such as hepatoma 36,37 and breast cancer 38
cells. Considering the biologic complexity of GSK-3 in vari-
ous normal cellular functions and the pathogenesis of various
cancers, we believe that this discrepancy is not surprising.

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Control
LiCI

B

■

14 21 28 35
Time (days)

42

49

o
£

3

2.5

1.5

0.5

Control

LiCI

Figure 5 In vivo validation of tumor growth inhibition by LiCI treatment.

Notes: After palpable tumors generated by SKOV3 cells developed, ip injections of LiCI (340 mg/kg) were done every 2 days for a total of seven treatments, the tumors
were measured, and the tumor volume was calculated once a week (A); The growth of the LiCI-treated tumors was slowed compared with the control; Data are means ±
SD (n-4); *P<0.05, # P<0.0 1 versus control; Tumor specimens dissected from the nude mice xenografted with SKOV3 cells at the end of the study (B); Comparison of tumor
weight between LiCI group and control (C); Data are means ± SD (n— 4); *P<0.05 versus control.
Abbreviations: ip, intraperitoneal; LiCI, lithium chloride; SD, standard deviation.

Recently, Cao et al 8 and Hilliard et al 10 showed GSK-3 as
a potential therapeutic target in ovarian cancer. Nevertheless,
the biological significance of GSK-3 in paclitaxel-resistant
ovarian cancer cells has not been evaluated. Our study revealed
that pharmacological inhibitors of GSK-3 can reduce viability

of SKOV3 and SKOV3-TR30 cells, a paclitaxel-resistant cell
subline derived from SKOV3. We also found that genetic
depletion of either GSK-3a or (3 isoforms can significantly
decrease viability of paclitaxel-sensitive and -resistant ovar-
ian carcinoma cells. Although most of the previous studies

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OncoTargets and Therapy 2014:7

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Aberrant activation of GSK-3 in ovarian cancer

have not focused on the significance of GSK-3 a, a role for
GSK-3a in acute myeloid leukemia has been demonstrated. 39
Our results indicate that both GSK-3 isoforms may contribute
to regulate cell viability in ovarian carcinoma.

To validate the in vitro findings of growth inhibition in
an in vivo situation, nude mice were implanted with SKOV3
cells. The dose of LiCl was chosen based on previous reports
evaluating subcutaneously grafted thyroid cancers. 28 Our
study showed that treatment of LiCl in mice bearing tumor
xenograft could significantly slow tumor growth compared
with the saline control. This finding was in agreement with
a previous study, although Cao et al mixed LiCl and tumor
cells together before subcutaneous injection. 8 The safety of
lithium ions has been established as a result of its use in the
treatment of psychotic diseases for over 60 years. Thus, the
possibility that LiCl could be a viable treatment for ovarian
cancer is exciting.

In summary, we have shown that overexpression and
aberrant activation of GSK-3 may contribute to progression
of ovarian cancer and have a potential role as independent
prognostic factors in EOC patients, and that inhibition of
GSK-3 may be a potential therapy for ovarian cancer. Further
studies are warranted to confirm and expand these findings,
including screening more reasonable administration sched-
ules of GSK-3 inhibitor/chemotherapy combination.

Acknowledgments

This work was supported by the Natural Science Foundation of
Zhejiang Province, People's Republic of China (LY12H16023)
and the National Natural Science Foundation of China
(30901591).

Disclosure

The authors report no conflicts of interest in this work.

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